The invention is based on the identification of aminopeptidase N (APN) as the receptor for F4 fimbriae of enterotoxigenic E. coli (ETEC). ETEC is an important cause of bacterial diarrheal illness. It is the leading cause of travelers' diarrhea and a major cause of diarrheal disease in underdeveloped nations. Based on the observation that oral administration of F4 fimbriae induces a protective intestinal mucosal immune response against a subsequent challenge with F4 ETEC, and the observation that the mechanism of F4 fimbriae endocytosis is clathrin-mediated, the present invention provides the characterization of APN as a target:                In an in vitro assay to screen for molecules that are capable to mimic the clathrin-mediated F4 endocytosis;        In an in vitro assay to screen for molecules that are capable to modulate the binding of F4 fimbriae with APN;        In the development of a carrier for the delivery of antigens/therapeutics, i.e. immunomodulators to the intestinal submucosa or the intestinal mucosa-associated lymphoid tissue, wherein said carrier comprises an APN specific target molecule that mimics the clathrin-mediated F4 endocytosis.        
The use of the carriers thus identified or the treatments thus identified, in a method of inducing an antigen-specific intestinal mucosal immune response, and/or in the treatment of bacterial diarrhea, is a further aspect of the present invention. Enterotoxigenic Escherichia coli, or ETEC, is an important cause of bacterial diarrheal illness in man and animal. Infection with ETEC is the leading cause of travelers' diarrhea and a major cause of diarrheal disease in underdeveloped nations, where it can be life-threatening among children (Ratchtrachenchai 2004). In pigs, ETEC can express five types of fimbriae; F4, F5, F6, F18 and F41 of which F4 is the most frequent (80%) and is involved in diarrhea and mortality (39%) in neonatal, suckling and newly weaned piglets (Conzelman 2000).
Susceptibility to F4 E. coli adhesion is dominantly inherited in the host and conferred by specific receptors on the brush border of enterocytes in the small intestine (F4 receptor positive). In homozygous resistant pigs (F4 receptor negative), no adhesion of F4 fimbriae is observed. Three antigenic variants have been identified namely F4ab, F4ac and F4ad of which F4ac is by far the most common type. Interestingly, oral immunization with soluble F4ac fimbriae has been reported to result in a protective immune response against a challenge with F4ac ETEC (Van den Broeck 1999). In addition, oral administration of recombinant F4ac on itself is also able to induce an immune response and experiments using F4ac coupled to human serum albumin have demonstrated that F4ac has potential to serve as a carrier molecule to induce mucosal immune responses against coupled antigens (Verdonck 2005). We found that orally administered F4ac is endocytosed by villous enterocytes, follicle-associated enterocytes and M cells in the epithelial brush border, whereafter transcytosis occurred into the lamina propria and dome regions of the jejunal and ileal Peyer's patches (Snoeck 2008). Subsequent uptake and presentation of F4ac by antigen presenting cells could explain its capacity to induce a mucosal immune response (Snoeck 2008). This implies that targeting selected antigens to one or more of the F4ac receptors may have potential to elicit efficient mucosal immune responses against these antigens. Intestinal mucin-type glycoprotein 1 and 2 have been identified as receptors for F4ac (Francis 1998, Erickson 1992), but these have not been reported to initiate transcytosis or to induce an efficient immune response, and are therefore not likely to represent the F4ac receptors that are involved in transcytosis and mucosal immune responses.
It has thus been an object of the present invention to identify the F4ac receptor involved in triggering the observed intestinal mucosal immune response mentioned hereinbefore. The identification of this receptor and the mechanism of F4 endocytosis provides the possibility of targeting said receptor and the F4 endocytosis mechanism in the development of a carrier for delivering an agent across the mucosal barrier.